Ultrasonographic and endocrine aspects of follicle deviation, and acquisition of ovulatory capacity in buffalo (Bubalus bubalis) heifers

The objectives of this study were to determine the interval from ovulation to deviation and the diameter of the dominant (DF) and largest subordinate (SF) follicles at deviation in buffalo (Bubalus bubalis) heifers. Two methods of evaluation (observed vs. calculated) were used. FSH and LH profiles encompassing follicle deviation (Experiment 1), and the follicular diameter when the DF acquired ovulatory capacity (Experiment 2) were also determined. The time of deviation and the diameter of the DF and the largest SF at deviation did not differ between observed and calculated methods. Overall, follicle deviation occurred 2.6 ± 0.2d (mean ± SEM) after ovulation, and the diameters of the DF and SF at deviation were 7.2 ± 0.2 and 6.4 ± 0.2mm, respectively. No changes in plasma levels of FSH or LH were observed (P=0.32 and P=0.96, respectively). Experiment 2 was conducted in two phases according to the diameter of the DF during the first wave of follicular development at the time of LH challenge (25mg of pLH). In the first phase, follicles ranging from 5.0 to 6.0mm (n=7), 6.1 to 7.0mm (n=11), or 7.1 to 8.0mm (n=9) were used, and in the second phase, follicles ranging from 7.0 to 8.4mm (n=10), 8.5 to 10.0mm (n=10), or 10.1 to 12.0mm (n=9) of diameter were used. After the pLH treatment, the DF was monitored by ultrasonography every 12h for 48h. No ovulations occurred in heifers in the first phase. However, in the second phase, an effect of follicular diameter was observed on ovulation rate [7.0-8.4mm (0.0%, 0/10), 8.5-10.0mm (50.0%, 5/10), and 10.0-12.0mm (55.6%, 5/9)]. In summary, follicle deviation occurred 2.6d after ovulation in buffalo (B. bubalis) heifers, when the diameters of the DF and SF were 7.2 and 6.4mm, respectively. No significant changes in plasma concentrations of FSH or LH were detected. Finally, the acquisition of ovulatory capacity occurred when the DF reached 8.5mm in diameter.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Differential Distribution of Both IL-12Rβ Chains in the Plasma Membrane of Human T Cells

IL-12 is a cytokine that stimulates the expression of CD26, a T cell– and raft-associated ectopeptidase. IL-12 also enhances the interaction between CD26 and CD45RO, which removes the phosphatase CD45RO from raft microdomains. Since Janus kinases are known CD45 substrates, our hypothesis was that this relocation of CD45RO in nonraft areas of the membrane could be important to switch off the signaling via cytokine receptors, e.g., the IL-12 receptor (IL-12R). Accordingly, both IL-12R and CD45RO should be equally positioned in the cell membrane upon IL-12R ligation. However, there were no data available on the membrane distribution of IL-12R on human T cells. Working with phytohemagglutinin (PHA) lymphoblasts, we tried to fill that gap. The high-affinity IL-12R is made of two chains: IL-12Rβ1 and IL-12Rβ2. Using flow cytometry, Western blot and confocal microscopy, we obtained data suggesting that IL-12Rβ1 is mainly associated to phospholipid-rich membrane areas, a location even enhanced upon IL-12 incubation of PHA blasts. Instead, IL-12Rβ2 is found more segregated into membrane rafts, which could explain why two IL-12-triggered events, T-cell proliferation and ERK1/2 activation, are both methyl-β-cyclodextrin-sensitive events. Ligation of IL-12R with IL-12 seems to induce a partial enrichment of IL-12Rβ2 in phospholipid-rich areas, where according to our data IL-12Rβ1 is already present. Therefore, although new data will be required, the present results support the initial hypothesis.     

Genotypic and Phenotypic Characterisation of Enteroaggregative Escherichia coli from Children in Rio de Janeiro,Brazil

Enteroaggregative Escherichia coli (EAEC) is a significant cause of diarrhoeal illness in both children and adults. Genetic heterogeneity and recovery of EAEC strains from both healthy and diseased individuals complicates our understanding of EAEC pathogenesis. We wished to establish if genetic or phenotypic attributes could be used to distinguish between strains asymptomatically colonising healthy individuals and those which cause disease. Genotypic screening of a collection of twenty four EAEC isolates from children with and without diarrhoea revealed no significant differences in the repertoire of putative virulence factors present in either group of strains. In contrast, EAEC strains from phylogroup A were more strongly associated with asymptomatic groups whereas strains from phylogroup D were more associated with cases of diarrhoea. Phenotypic screening revealed no differences in the ability of strains from either cohort of children to form biofilms, to adhere to and invade cells in tissue culture or to cause disease in the Caenorhabditis elegans model of infection. However, the latter assay did reveal significant reduction in nematode killing rates when specific virulence factors were deleted from human pathogenic strains. Our results suggest that current models of infection are not useful for distinguishing avirulent from pathogenic strains of EAEC but can be useful in studying the effect of specific virulence factors.     

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

Background  

MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species.     

Conservation study of an endangered stingless bee (Melipona capixaba—Hymenoptera: Apidae) with restricted distribution in Brazil

Melipona capixaba, popularly known as “uruçu preta”, is a stingless bee restricted to the mountainous Atlantic Rainforest areas of Espírito Santo State, Brazil. Due to the endemism and small population size, this species discovered in 1994 is now considered “vulnerable to extinction”. Using ISSR, PCR–RFLP and microsatellites markers, we studied the genetic variability and structure of M. capixaba from 88 colonies collected throughout the distribution area of the species within Espírito Santo State. The microsatellite, ISSR and mitochondrial haplotype analyses showed that M. capixaba has low genetic variability compared to other insect species. The molecular analyses also indicated a high genetic similarity among the M. capixaba samples, with no clear pattern of structuring. The analyses of molecular variance results indicated that most of the total genetic variation in M. capixaba was explained by the genetic diversity within local populations. Results suggest that the analyzed samples could be treated as a single population for preservation purposes. Thus, given its endemism, local adaptation and low number of natural colonies, efforts for the conservation of M. capixaba should focus on preservation and increasing the number of colonies in the wild, so that M. capixaba can support constant captures and the effects of habitat deforestation in Espírito Santo State.